Vitis 54 (Special Issue), 169171 (2015)

Resveratrol concentration in Vranac wines

B. RADOVI1), V. TEEVI1), V. KODULOVI2) and V. MARA2)
1)
Chemical Faculty, University of Belgrade, Belgrade, Serbia
2)
Biotechnical Faculty, University of Montenegro, Podgorica, Montenegro

Summary wine constituents. Phenolic compounds are an important

Red wine is the main source of resveratrol. It oc-
curs in both, isomeric (trans- and cis-) and in its gluco-
sides forms. The grapevine variety, climate, conditions
and duration of wine storage, and phytosanitary condi-
tions of grape are some of the factors that influence
content of resveratrol. This study analyzed trans- and
cis-resveratrol isomers concentrations and trans-res-
veratrol-3-O-glucoside and cis-resveratrol-3-O-gluco-
side concentrations among three clone candidates and
population of autochthonous Montenegrin grapevine
variety Vranac. Data from period of three years were
collected and analyzed with UPLC-MS/MS. Results
showed no statistically significant difference (p = 0.169)
in concentration of resveratrol among three clone can-
didates and population of grapevine variety Vranac.
K e y w o r d s : clones; UPLC-MS/MS; stilbenes; grape-
vine populations.
Introduction
Vranac is an autochthonous grapevine variety of
Montenegro, with a significant importance for the viticul-
ture and winemaking industries, and for the Montenegrin
economy in general. Vranac grapes have been used for
the production of premium quality red wines. However,
since 2004, researchers worked on developing clones
with improved agro-biological, economical and techno-
logical characteristics. As part of this project three year
(2010-2012) research was conducted in order to determine
the concentration of resveratrol in wine of three putative
Vranac clones.
Resveratrol is a member of the stilbene family and it
can exist in cis- or trans- configurations (trans-3, 5, 4-
trihydroxystilbene), or as glucosylated forms called piceid
(resveratrol-3-O-beta-mono-D-glucoside) (Figure). Both
trans- and cis-resveratrol are known as phenolic bioactive
group of substances in wine that contribute to several sen-
sory characteristics, such as color, flavor and astringency
(FRMONT 2000). Furthermore, it has been reported that
phenols have multiple biological effects, such as antioxi-
dant activity, anti-inflammatory action, inhibition of plate-
let aggregation and antimicrobial activity (EKI et al.
2008).
Concentration of resveratrol in wine depends on mul-
tiple factors. It is known that resveratrol is synthesized in
the plant in response to infection caused by fungal diseases
such as Botrytis cinerea or due to abiotic stress such as
UV radiation excess. It is assumed that the UV-B radiation
stimulates the production of enzymes responsible for the
synthesis of stilbene, in order to protect berry cells from
UV-induced damages (LANGCAKE et al. 1976). Red wines
and grapes present higher levels of resveratrol than white
grape products (MORENO et al. 2008). Cis-isomer is pro-
duced by UV irradiation of the trans-isomer (MORENO et
al. 2008). It is generally absent or only slightly detectable
in grapes, but both isomers are present in variable amounts
in commercial wines (CVEJI at al. 2010).
Cis-resveratrol was not reported at first as a natural
constituent of grape berries (JEANDET et al. 1991, JEANDET
et al. 1995). However, later, MORENO et al. 2008, detected
cis-isomer in red grape skins. Moreover, cis-resveratrol
and its glucoside have been detected in wines. More like-
ly, cis-resveratrol is derived from its trans-isomer during
vinification (CVEJI et al. 2010) even if the mechanisms of
its formation during winemaking are still unclear (DOUR-
TOGLOU et al. 1999).
Piceid is a derivative of resveratrol. The average con-
tent of piceid was found ten times higher than that of res-
veratrol in the root of Polygonum cuspidatum and in red
wine (DAN et al. 2013). Additionally, piceid was the most
abundant form of resveratrol in nature (REGEV-SHOSHA-
NI et al. 2003). A number of studies have suggested that
piceid may also have anti-carcinogenic effects (SOLEAS
et al. 2001), inhibition of platelet aggregation (CHUNG

Figure: Structures of free and glycosylated trans- and cis- resveratrol forms.

Correspondence to: Dr. B. RADOVIC, Jelene Bali 18, 81000 Podgorica, Montenegro. E-mail: radovicbojan081@gmail.com
170 B. RADOVI et al.

et al. 1992, ORSINI et al. 1997), and anti-oxidation activity
(BROHAN et al. 2011). This work aimed at developing new
Vranac clones with high concentrations in resveratrol and
its glucoside. The goal of the study was to determine con-
centrations of stilbenes (trans- and cis-resveratrol and its
piceid) in three developed clones and to statistically test
the difference in resveratrol concentrations between clones
and population of Vranac variety.
Material and Methods
Wines from the Vranac population and its three pu-
tative clones were produced in the experimental winery
of Plantaze Co. Standard vinifications were performed in
Ganimede tanks. Potassium meta-bisulfite (Agroterm
KFT, Hungary) was added (8 g100 kg-1 of grapes). All en-
zymes, wine yeast, lactic acid bacteria and yeast nutrients
were purchased from Lallemand, Australia. In more details,
Enoferm BDX (30 ghL-1) ; Go-ferm protect (30 ghL-1);
Lalvin EX-V (2 g100 kg-1); Fermaid E (25 ghL-1) were
added during fermentation. For malolactic fermentation
commercial lactic acid bacteria Lalvin VP41 was used for
inoculation.
The analyses were performed on four wine samples
produced in three years (2010, 2011, 2012).Trans-resvera-
trol standard was purchased from Sigma Chemical Co. (St.
Louis, MO). Cis-resveratrol is not commercially available
and was therefore obtained through UV-photoisomeriza-
tion (280-310 nm, for 10 h) of a standard solution (100
mgL-1) of trans-resveratrol. Under these conditions, 80 %
of the trans-resveratrol was converted into the cis-isomer.
U P L C M S / M S a n a l y s e s : Analyses of res-
veratrol was carried out on Waters Acquity UPLC H-Class
(WAT-176015007) equipped with a quaternary pump
Waters Quaternary Solvent Menager; an injector Waters
Sample Menager-FTN (Flow Through Needle); a column
compartment with a ZORBAX Eclipse XDB C18 column
(150 4,6 mm; 5 m); a Photodiode Arraydetector Wa-
ters 2998 PDA and a mass detector Waters TQ (Tandem
Quadrupole, WAT-176001263). For data acquisition and
processing, the software MassLynx V4.1 was used.
The mobile phase consists of 0.2 % fumaric acid in
H2O (A) and acetonitrile (B). The chromatographic separa-
tion was performed using a six stage linear gradient: from
5 % to 16 % of B in 20 min, from 16 to 40 % of B in 8 min,
from 40 % to 70 % of B in 4 min, from 70 to 100 % of B
in 4 min, and at 100 % of B during 9 min, and back to 5 %
of B in 9 min, with a total flow rate of 0.7 mLmin-1. The
total gradient time was 55 min. The separation temperature
was set up at 25 C. UV-visible spectra were recorded in
the wavelength range from 190 nm to 600 nm. Injection
volume was 10 L. MS Scan standard resveratrol was re-
corded in ESI- and ESI+ mode. The transition m/z 229
107 was monitored in ESI+ MRM mode (Multiple Reaction
Monitoring Mode) under the following conditions: capil-
lary voltage at 3.5 kV, the cone voltage 34 V, collision ener-
gy of 24 eV, the source temperature of 150 C, desolvation
temperature of 450 C, flow of desolvation gas 800 Lh-1.
The samples were analyzed without prior preparation: they
were only filtered through a filter Econofilter 25/0.45 m.
Calibration graphs were obtained by plotting the peak
area against the concentration. Five standards of trans- and
cis-resveratrol were used covering the range 0.05-5 mgL-1.
Concentration for trans- and cis-piceid were obtained from
calibration graphs for trans- and cis-resveratrol respective-
ly. The resveratrol concentration among the 4 Vranac-
types (Clone 1, Clone 2, Clone 3 and Population) over
three years were compared and statistically tested using re-
peated measures ANOVA (p = 0.05). SPSS 15.0 software
was used for statistical analysis.
Results and Discussion
The results of resveratrol concentration variabil-
ity among the 4 Vranac-types, obtained from the wine
analysis, are summarized in the Table. Results showed that
the highest average concentration of trans-piceid (1.123
mgL-1) was found in C lone III, while the highest aver-
age concentration of cis-piceid (1.193 mgL-1) was found in
the Population sample. The highest average concentration
of trans-resveratrol (0.600 mgL-1) among all the samples
was found in the Population sample. The highest average
concentration of total resveratrol (3.119 mgL-1) was found
in Clone I.
Resveratrol concentrations among the 4 Vranac-types
were statistically tested using repeated measure analysis
of variance (rANOVA) at p = 0.05. Results showed that
there was no statistically significant difference in total con-
centration of resveratrol among the tested Vranac-types
(F(3,6) = 2.373, p < 0.169). Similarly, results showed that

Ta b l e

Concentrations (mean and st. dev.) of trans-resveratrol (mgL-1), cis-resveratrol (mgL-1), c-resveratrol-3-O-glucoside
(mgL-1) and cis-resveratrol-3-O-glucoside (mgL-1) in wine of population of 'Vranac' variety and its putative clones in
period 2010- 12 in Plantaze A.D. winery, Podgorica, Montenegro

Resveratrol (mgL-1) Population putative Clone I putative Clone II putative Clone III
trans-resveratrol 0.600 (0.129) 0.485 (0.084) 0.414 (0.062) 0.457 (0.107)
cis-resveratrol 0.380 (0.058)a 0.318 (0.049) 0.295 (0.084) 0.190 (0.174)
trans-resveratrol-3-O-glucoside 0.834 (0.344) 1.123(0.196) 0.837 (0.237) 1.179 (0.067)
cis-resveratrol-3-O-glucoside 0.506 (0.162)a 1.193 (0.323)b 0.870 (0.090)ab 0.988 (0.030)b
Total 2.320 (0.317) 3.119 (0.596) 2.416 (0.180) 2.914 (0.317)
In the same line, when reported, means followed by the same letter are not statistically different.
 Resveratrol concentration in Vranac wines
there was no statistically significant difference in concen-
tration of trans-resveratrol among the tested Vranac-types
(F(3,6) = 2.730, p < 0.169) either.
The only statistically significant difference found
concerned in the variability in cis-resveratrol concentra-
tion among the tested Vranac-types (F(3,6) = 29.876,
p < 0.032). However, Tuckey post-hoc tests (with Bonfer-
roni adjustment for multiple comparisons) showed that
neither cis-resveratrol concentration among Vranac-types
was statistically significant at p = 0.05. Finally, results
showed that there was a statistically significant difference
in concentration of cis-resveratrol-3-O-glucoside among
the tested Vranac-types (F (3,6) = 70.087, p < 0.033).
Tuckey post-hoc tests (with Bonferroni adjustment for
multiple comparisons) showed that a statistically signifi-
cant difference in cis-resveratrol-3-O-glucoside concentra-
tion among the tested Vranac-types was found between
the Population wines and Clone III pair (at p = 0.024),
and between Clone II and Clone III pair (at p = 0.039).
Similarly, the results showed that there was a statistically
significant effect of concentration of cis-resveratrol among
the tested Vranac-types (F(3,6) = 29.876, p < 0.032).
However, Tuckey post-hoc tests (with Bonferroni adjust-
ment for multiple comparisons) showed that cis-resveratrol
concentration among groups was not statistically signifi-
cant at p = 0.05.
The study was conducted with the primary focus being
directed on the determination of trans-resveratrol and cis-
resveratrol levels and their piceid in Vranac wines. While
there are numerous factors that could have impacted the
total content of resveratrol (such as environmental and cli-
mate conditions), results indicate that Vranac clones have
the same capacity to produce resveratrol concentrations in
wine as the Vranac population.
171
References
BROHAN, M.; JERKOVIC, V.; COLLIN, S.; 2011: Potentiality of red sorghum
for producing stilbenoid-enriched beers with high antioxidant activ-
ity. J. Agric. Food Chem. 59, 4088-4094.
CHUNG, M. I.; TENG, C. M.; CHENG, K. L.; KO, F. N.; LIN, C. N.; 1992:
An antiplatelet principle of Veratrum formosanum. Planta Med. 58,
274-276.
CVEJI, J.; EKI, S.; PETROVI, A.; ATANACKOVIC, A.; JOVI, S.; BRCESKI,
I.; GOJKOVI-BUKARICA, L.; 2010: Determination of trans- and cis-
Resveratrol in Serbian commercial wines. J. Chromatogr. Sci. Vol.
48, 229-234.
DAN, S.; YING, C.; MIAO, L.; DAOZHOU, L.; HAN, C.; BANGLE, Z.; SIYUAN,
Z.; TIEHONG, Y.; QIBING, M.; 2013: Comparision of piceid and resver-
atrol in antioxidation and antiproliferation activities in vitro. PLoS
ONE 8, e54505.
EKI, S.; MILOSAVLJEVI, S.; VAJS, V.; JOVI, S., PETROVI, A.; NIKIEVI,
N.; MANOJLOVI, V.; NEDOVI, V.; TEEVI, V.; 2008: Trans- and
cis-resveratrol concentration in wines produced in Serbia, J. Serb.
Chem. Soc. 73, 1027-1037.
DOURTOGLOU, V. G.; MAKRIS, D. P.; BOIS-DOUNAS, D.; ZONAS C.; 1999:
Trans-resveratrol concentration in wines produced in Greece. J.
Food Comp. Anal. 12, 227-233.
FREMONT, L.; 2000: Biological effects of resveratrol. Life Sci. 66,
663-673.
JEANDET, P.; BESSIS R.; GAUTHERON, B.; 1991: The production of resvera-
trol (3.5.4-trihydroxystilbene) by grape berries in different develop-
mental stages. Am. J. Enol. Vitic. 42,41-46.
JEANDET, P.; BESSIS, R.; MAUME, B. F.; MEUNIER, P.; PEYRON, D.; TROLLAT,
P.; 1995: Effect of enological practices on the resveratrol isomer
content of wine. J. Agric.Food Chem. 43, 316-319.
JEANDET, P.; BESSIS, R.; SBAGHI, M.; MEUNIER, P.; TROLLAT, P.; 1995: Res-
veratrol content of wines of different ages: Relationship with fungal
disease pressure in the vineyard. Am. J. Enol. Vitic. 46, 1-4.
LANGCAKE, P.; PRYCE, R. J; 1976: The production of resveratrol by Vitis-
vinifera and other members of the Vitacee as a response to infection
or injury. Phys. Plant Pathol. 9,77-86.
MORENO, M.; CASTRO, E.; FALQU, E.; 2008: Evolution of trans- and
cis- resveratrol content in red grapes (Vitis vinifera L. cv Menci,
Albarello and Merenzao) during ripening. Eur. food Res. Technol.
227,667-674.
ORSINI, F.; PELIZZONI, F.; VEROTTA, L.; ABURJAI, T.; ROGERS C.B.; 1997: Iso-
lation, synthesis, and antiplatelet aggregation activity of resveratrol
3-O-beta-Dglucopyranoside and related compounds. J. Nat. Prod.
60, 1082-1087.
REGEV-SHOSHANI, G.; SHOSEYOV, O.; BILKIS, I.; KEREM, Z.; 2003: Glyco-
sylation of resveratrol protects it from enzymic oxidation. Biochem.
J. 374, 157-163.
SOLEAS, G. J.; GOLDBERG, D. M.; GRASS, L.; LEVESQUE, M.; DIAMANDIS, E.
P.; 2001: Do wine polyphenols modulate p53 gene expression in hu-
man cancer cell lines? ClinBiochem 34, 415-42.